Abstract
In Escherichia coli, FtsK is a large integral membrane protein that coordinates chromosome segregation and cell division. The N-terminal domain of FtsK (FtsKN) is essential for division, and the C terminus (FtsKC) is a well characterized DNA translocase. Although the function of FtsKN is unknown, it is suggested that FtsK acts as a checkpoint to ensure DNA is properly segregated before septation. This may occur through modulation of protein interactions between FtsKN and other division proteins in both the periplasm and cytoplasm; thus, a clear understanding of how FtsKN is positioned in the membrane is required to characterize these interactions. The membrane topology of FtsKN was initially determined using site-directed reporter fusions; however, questions regarding this topology persist. Here, we report a revised membrane topology generated by site-directed fluorescence labeling. The revised topology confirms the presence of four transmembrane segments and reveals a newly identified periplasmic loop between the third and fourth transmembrane domains. Within this loop, four residues were identified that, when mutated, resulted in the appearance of cellular voids. High resolution transmission electron microscopy of these voids showed asymmetric division of the cytoplasm in the absence of outer membrane invagination or visible cell wall ingrowth. This uncoupling reveals a novel role for FtsK in linking cell envelope septation events and yields further evidence for FtsK as a critical checkpoint of cell division. The revised topology of FtsKN also provides an important platform for future studies on essential interactions required for this process.
Highlights
FtsK is an essential membrane protein that links chromosome segregation and cell division in bacteria
The role of FtsK as a potential checkpoint of cell division relies upon its ability to efficiently couple both chromosome segregation and septation in bacteria
The precise role that FtsK plays in cell division is unknown, evidence suggests that multiple regions along FtsK connect essential components of the divisome, including the Z-ring and peptidoglycan synthesis machinery, and that modulation of these interactions may delay septation to allow for proper DNA segregation [17, 25, 26]
Summary
FtsK is an essential membrane protein that links chromosome segregation and cell division in bacteria. In 2010, Dubarry et al [17] speculated on the functional link between FtsK in cell division and DNA segregation and suggested a mechanism of action that highlights its potential role as a checkpoint of bacterial cell division This proposed mechanism emphasizes an inverse relationship between septum formation and DNA translocation that is mediated by allosteric changes in FtsK and modulation of protein-protein interactions within the N-terminal domain. FtsKN contains four transmembrane ␣-helices connected by a moderately sized periplasmic and cytoplasmic loop, as well as a third periplasmic segment containing a single amino acid residue [38] Because this topology mapping technique relies on severe truncation of the target protein, we must assume these truncated and usually inactive constructs maintain the same native topology as the full integral membrane portion of the protein [39]. A series of residues in the newly identified periplasmic loop were identified that, when mutated, uncoupled invagination of the inner and outer membranes and resulted in visible voids in the cellular material
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